Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
  • C12P17/185—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system
  • C12P17/186—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system containing a 2-oxo-thieno[3,4-d]imidazol nucleus, e.g. Biotin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales

Definitions

• This invention relates to a process for the production of d-biotin by fermentation.
• d-Biotin is one of the essential vitamins for the nutrition of animals, plants and microorganisms, and is very important as a medicine or food additive.
• the present invention enables the production of d-biotin by fermentation to occur in high yield.
• the present invention enables large amounts of d-biotin to be accumulated. It has been found that microorganisms belonging to the genus Kurthia and which are resistant to biotin antimetabolites, e.g. acidomycin (hereinafter referred to as ACM), 5-(2-thienyl)-valeric acid (TVA), ⁇ -methyl dethiobiotin (MeDTB), 2-methyl ACM, amiclenomycin, bisnorbiotinol and mixtures thereof, are capable of accumulating large amounts of d-biotin in the culture broth and that the d-biotin can be recovered therefrom in good purity.
• ACM acidomycin
• TVA 5-(2-thienyl)-valeric acid
• MeDTB ⁇ -methyl dethiobiotin
• 2-methyl ACM amiclenomycin
• bisnorbiotinol bisnorbiotinol and mixtures thereof
• d-biotin by the present invention in about 5 to 200 times higher yields than those achieved with known methods utilizing strains belonging to the genus Bacillus or Serratia.
• a process for producing d-biotin which comprises cultivating a microorganism belonging to the genus Kurthia, which microorganism is resistant to biotin antimetabolites and capable of producing d-biotin in a medium under aerobic conditions, and separating the resulting d-biotin from the fermentation broth.
• Microorganisms which can be used in accordance with the present invention include all the d-biotin-producing microorganisms which belong to the genus Kurthia see Bergey's Manual of Systematic Bacteriology, 9 th ed., vol. 2, 1255 (1984)! and are resistant to biotin antimetabolites, such as those mentioned above.
• the above-mentioned microorganisms can be obtained efficiently by treating strains belonging to the genus Kurthia as parent strains with a mutagen, e.g. N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as NTG), ethyl methane sulfonate, acridine orange, UV- or X-rays.
• NTG N-methyl-N'-nitro-N-nitrosoguanidine
• Any strain belonging to the genus Kurthia can be used as a parent strain for preparing the microorganisms used in accordance with the present invention.
• the microorganisms belonging to the genus Kurthia may be isolated from natural sources or may be purchased from culture collections. Kurthia sp. 533-6 (DSM No. 9454) is preferably used for the present invention.
• the microorganisms which are capable of producing large amounts of d-biotin can be obtained by isolating a microorganism belonging to the genus Kurthia and being resistant to biotin antimetabolites, such as those mentioned above. For example, a mutant of Kurthia sp. 538-6 (DSM No. 9454) producing large amounts of d-biotin was isolated as described below.
• the content of d-biotin accumulated in the culture broth can be assayed by the turbidity method J. Microbiological Methods, 6, 237-245 (1987)! by means of Lactobacillus plantarum ATCC 8014 Proc. Soc. Exp. Biol. Med., 56, 95-98 (1944)!.
• the washed cells were spread onto agar plates of a d-biotin-free synthetic medium comprising 20 g of glycerol, 5 g of vitamin-free casamino acids (Difco), 2 g of K 2 HPO 4 , 1 g of KH 2 PO 4 , 0.5 g of MgSO 4 ⁇ 7H 2 O, 0.01 g of FeSO 4 ⁇ 7H 2 O, 0.01 g of MnSO 4 ⁇ 5H 2 O and 100 ⁇ g of thiamine ⁇ HCl in 1,000 ml of distilled water (hereinafter referred to as BM) with 100 ⁇ g/ml of ACM.
• BM thiamine ⁇ HCl in 1,000 ml of distilled water
• Kurthia sp. 538-KA 26 was cultured and the cells were treated with NTG in a similar manner as described in (1) above.
• the treated cells were cultured in a d-biotin-free synthetic medium composed of 20 g of glycerol, 3 g of (NH 4 ) 2 SO 4 , 2 g of K 2 HPO 4 , 1 g of KH 2 PO 4 , 0.5 g of MgSO 4 ⁇ 7H 2 O, 0.01 g of FeSO 4 ⁇ 7H 2 O, 0.01 g of MnSO 4 ⁇ 5H 2 O, and 100 ⁇ g of thiamine ⁇ HCl in 1,000 ml of distilled water (hereinafter referred to as MM) with 200 ⁇ g/ml of ACM and 200 ⁇ g/ml of TVA, and then transferred serially several times in a fresh medium with 200 ⁇ g/ml of ACM and 200 ⁇ g/ml of TVA at 2 to 3 day intervals.
• MM thiamine ⁇ HC
• the resulting enriched cultures were diluted and spread onto agar plates of MM containing 200 ⁇ g/ml of ACM and 200 ⁇ g/ml of TVA. After incubation, large colonies grown were inoculated onto the assay plates with Lactobacillus plantarum ATCC 8014 in a similar manner as described in (1) above. After incubation, the diameters of growth halos were measured. From the result, a colony producing the largest halo was obtained and designated Kurthia sp. 538-17H4.
• Kurthia sp. 538-17H4 was cultured and the cells were treated with NTG in a similar manner as described in (1) above.
• the treated cells were spread onto agar plates of BM containing 100 ⁇ g/ml of ACM. After incubation, the colonies were inoculated onto agar blocks of a production medium containing 0.5 ⁇ g/ml of d-dethiobiotin and 80 ⁇ g/ml of MeDTB in a plate. After incubation, the agar blocks were irradiated with UV light, and then transferred on the assay plates with Lactobacillus plantarum ATCC 8014. After incubation, the diameters of growth halos were measured. From the result, a colony producing the largest halo was obtained and designated Kurthia sp. 538-51F9.
• the process of the present invention is suitably effected by cultivating (incubating) the microorganism in a medium containing an assimilable carbon source, a digestible nitrogen source, inorganic salts and other nutrients necessary for the growth of the microorganism.
• an assimilable carbon source for example, glucose, fructose, starch, lactose, maltose, galactose, sucrose, dextrin, glycerol or millet jelly, preferably glycerol or glucose
• a nitrogen source for example, peptone, soybean powder, corn steep liquor, meat extract, ammonium sulfate, ammonium nitrate, urea or a mixture thereof may be employed, preferably peptone.
• sulfates hydrochlorides or phosphates of calcium, magnesium, zinc, manganese, cobalt and iron may be employed.
• Particularly suitable as inorganic salts are monopotassium phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate.
• conventional nutrient factors or an antifoaming agent such as animal oil, vegetable oil or mineral oil can also be added.
• the pH of the culture medium is conveniently about 5.0 to 9.0, preferably 6.5 to 7.5.
• the cultivation temperature is suitably about 10 to 40° C., preferably 26 to 30° C.
• the cultivation time may be about 1 to 10 days, preferably 2 to 7 days, most preferably about 2 to 4 days (48 to 96 hours). In the cultivation, aeration and agitation usually give favorable results.
• the d-biotin produced may be separated from the culture broth and purified.
• a process generally used for extracting a certain product from the culture broth may be applied by utilizing various properties of d-biotin.
• the cells are removed from the culture broth, the desired substance in the filtrate is absorbed on active carbon, then eluted and purified further with an ion exchange resin.
• the culture filtrate is applied directly to an ion exchange resin and, after the elution, the desired product is recrystallized from a mixture of alcohol and water.
• the microorganisms used according to the present invention include all the strains resistant to ACM, TVA, MeDTB, 2-methyl ACM, amiclenomycin, bisnorbiotinol, or mixtures thereof belonging to the genus Kurthia and being capable of producing d-biotin.
• particularly preferred strains are Kurthia sp. 538-KA 26, Kurthia sp. 538-17H4, Kurthia sp. 538-51F9 and Kurthia sp. 538-2A13, which were deposited at the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) in Braunschweig, Germany under DSM Nos. 10609, 10608, 10610 and 10607, respectively, on Mar. 26, 1996.
• DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
• the d-biotin productivity of each colony grown was determined by the agar plate method containing Lactobacillus plantarum ATCC 8014 as described below. Seven hundred and fifty colonies were transferred with toothpicks onto the assay plates containing Lactobacillus plantarum ATCC 8014. After incubation overnight at 37° C., the diameters of growth halos, which depended on the amount of d-biotin, were measured. As a result, Kurthia sp. 538-KA 26 (DSM No. 10609), producing the largest halo, was obtained.
• d-biotin was assayed by the turbidity method with Lactobacillus plantarum ATCC 8014 as described below.
• the supernatant and standard solutions of d-biotin (0-10 mg per liter) were diluted 1.25-10 -3 in distilled water. Fifty microliters of the diluted solution and 5 ml of distilled water were added to tubes in this order. After autoclaving at 120° C. for 10 minutes, 5 ml of the assay medium for d-biotin (Nissui Co.
• the cells of strain 538-KA 26 were treated with NTG, and the cells resistant to ACM and TVA were enriched as described below.
• the washed cells after NTG treatment were inoculated into tubes containing 5 ml of BM medium with 100 ⁇ g/ml of ACM, and the tubes were shaken on a reciprocal shaker (285 rpm) at 28° C. After overnight incubation, the culture broth was inoculated to give a turbidity at 600 nm of approximately 0.03 into a tube containing 5 ml of MM with 200 ⁇ g/ml of ACM and 200 ⁇ g/ml of TVA. The tubes were shaken at 28° C.
• the culture broth was transferred into a flask containing 50 ml of the production medium composed of 4% glycerol, 4.5% proteose peptone, 0.1% KH 2 PO 4 , 0.05% MgSO 4 ⁇ 7H 2 O, 0.05% FeSO 4 ⁇ 7H 2 O, 0.001% MnSO 4 ⁇ 5H 2 O and one drop of antifoam CA-115 (Nippon Yushi Co. Ltd., Japan), and then the flask was shaken at 28° C. After cultivation for 72 hours, the content of d-biotin in the supernatant of the culture broth was assayed by the turbidity method with Lactobacillus plantarum ATCC 8014.
• the production medium composed of 4% glycerol, 4.5% proteose peptone, 0.1% KH 2 PO 4 , 0.05% MgSO 4 ⁇ 7H 2 O, 0.05% FeSO 4 ⁇ 7H 2 O, 0.001% MnSO 4 ⁇ 5H 2 O and
• d-Biotin was recovered from the culture broth of Kurthia sp. 538-17H4 (DSM No. 10608). The d-biotin concentration at each purification step was followed by the turbidity method with Lactobacillus plantarum ATCC 8014. One liter of the 72 hour-culture broth having d-biotin activity of 25.7 mg/L against Lactobacillus plantarum ATCC 8014 was centrifuged at 7,500 rpm for 10 minutes. The supernatant was applied to a column (3.6 ⁇ 42 cm) packed with 380 ml of charcoal (Wako Pure Chemical Industries., Co. Ltd., Japan).
• the column After washing with 500 ml of deionized water, the column was developed with 500 ml of the ethanolic ammonia solution composed of 474 ml of 50% ethanol and 26 ml of 25% ammonia.
• 500 ml of the ethanolic ammonia solution composed of 474 ml of 50% ethanol and 26 ml of 25% ammonia.
• the concentrate was applied to a column (2.6 ⁇ 38 cm) packed with 190 ml of Dowex 1 ⁇ 4 (formate form, 100-200 mesh, Dow Chemical Co. Ltd., U.S.A.), and then the column was washed with 200 ml of 0.05 M formic acid.
• the column was developed with 250 ml of the starting buffer of 0.1 M formic acid and followed by the linear gradient of 250 ml each of 0.1 and 0.35 M formic acid.
• One hundred and sixty milliliters of the eluent having d-biotin activity of 144.8 mg/L against Lactobacillus plantarum ATCC 8014 were concentrated under reduced pressure to obtain 17.6 mg of white powder.
• This was crystallized from a mixture of ethanol and water to obtain 15.1 mg of white needles having a melting point of 232° C.
• infrared spectrum and NMR spectrum of the sample coincided with those of authentic d-biotin (Sigma Co. Ltd., U.S.A.).
• Example 2 the cells of Kurthia sp. 538-17H4 (DSM No. 10608) were treated with NTG, and a MeDTB-resistant mutant was then screened by the method as described below. After the treated cells had been cultured overnight at 28° C. in BM medium with 100 ⁇ g/ml of ACM, the culture was serially diluted 10 -1 to 10 -7 in sterile saline and spread onto BM agar with 80 ⁇ g/ml of ACM.
• agar blocks of the production medium composed of 2% glycerol, 2% proteose peptone, 0.1% KH 2 PO 4 , 0.05% MgSO 4 ⁇ 7H 2 O, 0.05% FeSO 4 ⁇ 7H 2 O, 0.001% MnSO 4 ⁇ 5H 2 O and 1.5% agar supplemented with 0.5 ⁇ g/ml of d-dethiobiotin and 80 ⁇ g/ml of MeDTB in plates.
• Kurthia sp. 538-51F9 (DSM No. 10610) grown on BM agar with 100 ⁇ g/ml of ACM was cultured at 28° C. in a tube containing 5 ml of BM medium with 500 ⁇ g/ml of ACM. After overnight cultivation, the cells were harvested by centrifugation and suspended in the 10% glycerol solution.
• One milliliter of the cell suspension was transferred into a flask containing 50 ml of the production medium composed of 6% glycerol, 5.5% proteose peptone ,0.1% KH 2 PO 4 , 0.05% MgSO 4 ⁇ 7H 2 O, 0.05% FeSO 4 ⁇ 7H 2 O, 0.001% MnSO 4 ⁇ 5H 2 O and one drop of antifoam CA-115 (hereinafter referred to as FM 1), and then the flask was cultured at 28° C. After cultivation for 72 hours, the content of d-biotin in the supernatant of the culture broth was assayed by the turbidity method with Lactobacillus plantarum ATCC 8014.
• FM 1 antifoam CA-115
• a mutant more resistant to MeDTB was derived from Kurthia sp. 538-51F9 (DSM No. 10610).
• the cells of Kurthia sp. 538-51 F9 (DSM No. 10610) were treated with NTG, and then mutants resistant to 150 ⁇ g/ml of MeDTB were screened by the method similar to that described in Example 4 except for the concentration of MeDTB in agar blocks.
• Kurthia sp. 538-2A13 (DSM No. 10607), producing the largest halo, was thus obtained.
• Kurthia sp. 538-2A13 (DSM No. 10607) was cultured at 28° C. in a flask containing 50 ml of FM 1 medium. After cultivation for 24 hours, 5 ml of the sterile medium containing 40% glycerol and 20% proteose peptone were fed, and the cultivation was continued. After cultivation for 120 hours, the content of d-biotin in the supernatant of the culture broth was assayed by the turbidity method with Lactobacillus plantarum ATCC 8014. As the result, the supernatant contained 126 mg of d-biotin per liter. Kurthia sp. 538-2A13 (DSM No. 10607) produced about 3 times as much biotin as the parent strain, Kurthia sp. 538-51F9 (DSM No. 10610), did.
• Table 1 summarizes the d-biotin productivities of the mutants so far obtained.

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